Influx of extracellular calcium is critical for a number of vital cellular processes. Calcium influx is generally mediated by calcium channels, which are grouped into several families one of which is the T type calcium channel family. Pharmacological modulation of the T type calcium channel's function is tremendously important in the practice of medicine; for example, T type calcium channel inhibitors are in widespread use in the treatment of neurological diseases (e.g. epilepsy, petit mal seizure, absence seizure, neuropathic pain, and etc.) and cardiovascular diseases (e.g. hypertension, unstable angina, and etc.). For example, mibefradil, a T type calcium inhibitor, was clinically efficacious in treating hypertension and cardiac arrhythmia. Studies also suggest that T-type calcium channels may play an important role in age related macular degeneration. Recently, we have showed that the α1H and δ 25 isoforms of T type calcium channels are present in cancer cell lines and that novel chemical agents could be synthesized to block calcium entry via this channel thus inhibiting cancer cell proliferation.
Ca2+ entry is critical for cellular proliferation. Because unusually rapid proliferation is a hallmark of cancer, elucidation of the mechanism of Ca2+ entry is of scientific importance with potential clinical significance. However, the mechanism or mechanisms of Ca2+ entry in electrically non-excitable cells, which constitute most types of cancer, remain elusive. The majority of cancers arise from cell types considered “electrically non-excitable.” This is to distinguish the means of regulation of Ca2+ entry in these cells from those having action potentials, that is “electrically excitable” cells. In electrically excitable cells, an action potential opens voltage gated Ca2+ channels that admit the Ca2+ required for initiating events such as neurosecretion. In electrically non-excitable cells, the regulatory mechanism activating Ca2+ entry is unclear in part because of the uncertainty about the molecular mechanism by which Ca2+ entry occurs.
The two types of ion channels or transporters most commonly implicated in capacitative Ca+ entry are ICRAC and members of the Trp. These two channel types do not share many properties other than relative selectivity for Ca2+, albeit sometimes very weak, and lack of voltage dependent gating. It has been difficult, however, to tie these proteins' function to capacitative Ca+ entry. This may reflect a particular complexity of Ca2+ signaling in electrically non-excitable cells arising from participation of numerous channels and transporters in this process. It is also possible that the primary means of Ca2+ entry in electrically non-excitable cells has yet to be fully elucidated.
We have taken an alternative approach to dissecting the Ca2+ entry pathway in electrically non-excitable cells. We first took advantage of Ca+ entry blockade by Ni2+, as measured by fluorescence techniques (Merritt, J. E. and Rink, T. J. 1987. Regulation of cytosolic free calcium in fura-2-loaded rat parotid.acinar cells. J. Biol. Chem. 262:17362-17369; Merritt, J. E., Jacob, R., and Hallam, T. J. 1989. Use of manganese to discriminate between calcium influx and mobilization from internal stores in stimulated human neutrophils. J. Biol. Chem. 264:1522-1527; Skryma, R., Mariot, P., Bourhis, X. L., Coppenolle, F. V., Shuba, Y., Abeele, F. V., Legrand, G., Humez, S., Boilly, B., and Prevarskaya, N. 2000. Store depletion and store-operated Ca2+ current in human prostate cancer LNCaP cells: involvement in apoptosis. J. Physiol. (Lond.) 527 Pt 1:71-83), to identify compounds in the published literature with a similar ability. The structure/activity relationship of these known compounds was used to guide the synthesis of novel compounds with enhanced potency to block Ca2+ entry into and proliferation of several cancer cell lines. Administration of one of these compounds, TH-1177, significantly extended the lifespan of nude mice inoculated with human PC3 prostate cancer cells (Haverstick, D. M., Heady, T. N., Macdonald, T. L., and Gray, L. S. 2000. Inhibition of human prostate cancer proliferation in vitro and in a mouse model by a compound synthesized to block Ca2+ entry. Cancer Res 60:1002-1008). We show here that two representative compounds block the Ca2+ current through the heterologously expressed α1H isoform of T type Ca2+ channels and inhibit proliferation of HEK293 cells stably transfected with this isoform. These two compounds blocked the Ca2+ current and capacitative Ca2+ entry with similar potencies and identical stereoselectivity. Importantly, cell lines sensitive to our novel compounds express message for α1H, its δ 25 splice variant, or both while a cell line resistant to our compounds does not detectably express either message.
The library of compounds we have developed has apparently broad activity against cancer cell lines from various tissues. They act cytostatically and are equally potent at inhibiting hormone sensitive and insensitive breast and prostate cancer lines. We demonstrate here the likely target of their anti-proliferative activity. Taken together, these observations raise the possibility of directed chemical synthesis of compounds that inhibit Ca2+ entry into and thereby proliferation of cancer cells.